This invention relates to novel bicyclic compounds which inhibit platelet aggregation, pharmaceutical compositions containing the compounds and methods of using the compounds.
Platelet aggregation is believed to be mediated primarily through the fibrinogen receptor, or GPIIb-IIIa platelet receptor complex, which is a member of a family of adhesion receptors referred to as integrins. It has been found that frequently the natural ligands of integrin receptors are proteins which contain an Arg-Gly-Asp sequence. Von Willebrand factor and fibrinogen, which are considered to be natural ligands for the GPIIb-IIIa receptor, possess an Arg-Gly-Asp (RGD in single letter amino acid code) sequence in their primary structure. Functionally, these proteins are able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and thereby effect aggregation of platelets.
Fibronectin, vitronecfin and thromospondin are RGD-containing proteins which have also been demonstrated to bind to GPIIb-IIIa Fibronectin is found in plasma and as a structural protein in the intracellular matrix. Binding between the structural proteins and GPIIb-IIIa may function to cause platelets to adhere to damaged vessel walls.
Linear and cyclic peptides which bind to vitronectin and contain an RGD sequence are disclosed in WO 89/05150 (PCT US88/04403). EP 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation. Other linear and cyclic peptides, the disclosure of which are incorporated herein by reference, are reported in EP-A 0 341 915. However, the peptide like structures of such inhibitors often pose problems, such as in drug delivery, metabolic stability and selectivity. Inhibitors of the fibrinogen receptor which are not constructed of natural amino acid sequences are disclosed in EP-A 0 372,486, EP-A 0 381 033 and EP-A 0 478 363. WO 92/07568 (PCT/US91/08166) discloses fibrinogen receptor antagonists which mimic a conformational xcex3-turn in the RGD sequence by forming a monocyclic seven-membered ring structure. There remains a need, however, for novel fibrinogen receptor antagonists (e.g. inhibitors of the GPIIb-IIIa protein) which have potent in vivo and in vitro effects and lack the peptide backbone structure of amino acid sequences.
The present invention discloses novel bicyclic compounds including benzazepines and benzodiazepines, which are inhibitors of the GPIIb-IIIa receptor and inhibit platelet aggregation. Certain 5-phenyl-1,4-benzodiazepines are known as a class of drugs which affect the central nervous system, and have been used as anxiolytics. See Sternbach, L. H., J. Med. Chem., 22, 2 (1979). It has also been disclosed that certain 5-phenyl-1,4-benzodiazepines antagonize the effects of cholecystokinin. See Friedinger, Med. Res. Rev., 9, 271 (1989). Certain bicyclic compounds which have fibrinogen antagonist activity are disclosed in WO 93/08174 (PCT/US92/08788) and WO 93/00095 (PCT/US/92/05463).
In one aspect this invention is a bicyclic compound comprising a substituted six-membered ring fused to a substituted seven-membered ring as described hereinafter in formula (I).
This invention is also a pharmaceutical composition for inhibiting platelet aggregation or clot formation, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier.
This invention further comprises the use of a compound of formula (I) in the manufacture of a medicament for inhibiting platelet aggregation.
In another aspect, this invention provides a method for inhibiting reocclusion of an artery or vein in a mammal following fibrinolytic therapy, which comprises internally administering an effective amount of a fibrinolytic agent and a compound of formula (I). This invention is also a method for treating stroke, transient ischemia attacks, or myocardial infarction.
This invention discloses novel bicyclic compounds which inhibit platelet aggregation. The novel bicyclic compounds comprise a seven-membered ring fused to an aromatic six membered ring and having a nitrogen-containing substituent on the six membered ring and an aliphatic substituent, preferably containing an acidic moiety, on the seven membered ring. The fused 6-7 ring system is believed to interact favorably with the GPIIb-IIIa receptor and to orient the substituent sidechains on the six and seven membered rings so that they may also interact favorably with the receptor.
Although not intending to be bound to any specific mechanism of action, these compounds are believed to inhibit the binding of fibrinogen to the platelet-bound fibrinogen receptor GPIIb-IIIa, and may interact with other adhesion proteins via antagonism of a putative RGD binding site.
The compounds of this invention are compounds of formula (I): 
wherein
A1 is O, S, Nxe2x80x94R1 or CHR1;
A4 is Nxe2x80x94R4 or CHR4;
R2 is R7 or Qxe2x80x94C1-4alkyl, Qxe2x80x94C2-4alkenyl or Qxe2x80x94C2-4alkynyl substituted by R7;
R1, R4 and R5 are H, Qxe2x80x94C1-6alkyl, Qxe2x80x94C1-6oxoalkyl, Qxe2x80x94C2-6alkenyl, Qxe2x80x94C3-4oxoalkenyl, Qxe2x80x94C3-4oxoalkynyl, Qxe2x80x94C2-4alkynyl, C3-6cycloalkyl, Ar or Het, optionally substituted by one or more of R11;
Q is H, C3-6cycloalkyl, Het or Ar;
R6 is Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94;
R7 is xe2x80x94COR8, xe2x80x94COCRxe2x80x22R9, xe2x80x94C(S)R8, xe2x80x94S(O)mORxe2x80x2, xe2x80x94S(O)mNRxe2x80x2Rxe2x80x3, xe2x80x94PO(ORxe2x80x2), xe2x80x94PO(ORxe2x80x2)2, xe2x80x94B(ORxe2x80x2)2, xe2x80x94NO2 and Tet;
R8 is xe2x80x94ORxe2x80x2, xe2x80x94NRxe2x80x2Rxe2x80x3, xe2x80x94NRxe2x80x2SO2Rxe2x80x2, xe2x80x94NRxe2x80x2ORxe2x80x2, xe2x80x94OCRxe2x80x22C(O)ORxe2x80x2, xe2x80x94OCRxe2x80x22OC(O)xe2x80x94Rxe2x80x2, xe2x80x94OCRxe2x80x22C(O)NRxe2x80x22, CH3 or AA;
R9 is xe2x80x94ORxe2x80x2, xe2x80x94CN, xe2x80x94S(O)rRxe2x80x2, S(O)mNRxe2x80x22, xe2x80x94C(O)Rxe2x80x2C(O)NRxe2x80x22 or xe2x80x94CO2Rxe2x80x2;
R10 is H, C1-4alkyl or xe2x80x94NRxe2x80x2Rxe2x80x3;
R11 is H, halo, xe2x80x94OR12, xe2x80x94CN, xe2x80x94NRxe2x80x2R12, xe2x80x94NO2, xe2x80x94CF3, CF3S(O)rxe2x80x94, xe2x80x94CO2Rxe2x80x2, xe2x80x94CONRxe2x80x22, Qxe2x80x94C0-6alkyl-, Qxe2x80x94C1-6oxoalkyl-, Qxe2x80x94C2-6alkenyl-, Qxe2x80x94C2-6alkynyl-, Qxe2x80x94C0-6alkyloxy-, Qxe2x80x94C0-6alkylamino- or Qxe2x80x94C0-6alkyl-S(O)rxe2x80x94;
R12 is Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94C(O)NRxe2x80x22, xe2x80x94C(O)OR15, xe2x80x94S(O)mRxe2x80x2 or S(O)mNRxe2x80x22;
R15 is H, C1-6alkyl or Arxe2x80x94C0-4alkyl;
Rxe2x80x2 is H, C1-6alkyl, C3-7cycloalkyl-C0-4alkyl or Arxe2x80x94C0-4alkyl;
Rxe2x80x3 is Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2 or xe2x80x94C(O)OR15;
Rxe2x80x2xe2x80x3 is Rxe2x80x3 or AA2;
AA1 is an amino acid attached through its amino group and having its carboxyl group optionally protected, and AA2 is an amino acid attached through its carboxyl group, and having its amino group optionally protected;
U and V are absent or CO, CRxe2x80x22, C(xe2x95x90CRxe2x80x22), S(O)n, O, NRxe2x80x2, CRxe2x80x2ORxe2x80x2, CRxe2x80x2(ORxe2x80x3)CRxe2x80x22, CRxe2x80x22CRxe2x80x2(ORxe2x80x3), C(O)CRxe2x80x22, CRxe2x80x22C(O), CONRxe2x80x2, NRxe2x80x2CO, OC(O), C(O)O, C(S)O, OC(S), C(S)NRxe2x80x2, NRxe2x80x2C(S), S(O)nNRxe2x80x2, NRxe2x80x2S(O)n, Nxe2x95x90N, NRxe2x80x2NRxe2x80x2, NRxe2x80x2CRxe2x80x22, CRxe2x80x22NRxe2x80x2, CRxe2x80x22O, OCRxe2x80x22, Cxe2x89xa1C or CRxe2x80x2xe2x95x90CRxe2x80x2, provided that U and V are not simultaneously absent;
W is Rxe2x80x2Rxe2x80x2xe2x80x3Nxe2x80x94 or ;
Z is (CH2)t or Het;
m is 1 or 2;
n is 0 to 3;
q is 0 to 3;
r is 0 to 2;
s is 0 to 2; and
t is 0 to 2; or
pharmaceutically acceptable salts thereof.
In another embodiment, this invention is a compound of formula (II): 
wherein R1, R2, R4, R5 and R11 are as defined in for formula (I) and R6 is Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94, where U, V and Rxe2x80x2 are as defined for formula (I), R10 is H, C1-4alkyl or xe2x80x94NRxe2x80x2Rxe2x80x3, W is Rxe2x80x22N, H2NC(xe2x95x90NH), H2NC(xe2x95x90NH)NH or ; and Z is Ar, C3-7cycloalkyl, (CH2)t or Het. In particular, compounds of formula (II) where R1 is H, C1-4alkyl or C(O)Rxe2x80x2; R2 is CH2CO2H or CH2CH2CO2H, R4 is H, C1-6alkyl, C3-6cycloalkylC0-4alkyl or Arxe2x80x94C0-4alkyl; and Z is phenyl, a six-membered Het or (CH2)t, are suitable.
In yet another embodiment of this invention, compounds of formula (III) are provided 
wherein R2, R5 and R11 are as defined for formula (I), and R6 is or 
wherein E is N or CH, R20 is hydrogen, amino, mono or di-C1-4alkylamino, hydroxy or C1-4alkyl, and U is NRxe2x80x2CO, CONRxe2x80x2, (CH2)CO, CHxe2x95x90CH or Cxe2x89xa1C.
Also included in this invention are pharmaceutically acceptable addition salts, complexes or prodrugs of the compounds of this invention. Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo.
In cases wherein the compounds of this invention may have one or more chiral centers, unless specified, this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, such as 
and tautomers of guanidine-type groups, such as 
each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or locked in one form by appropriate substitution with Rxe2x80x2. The meaning of any substituent at any one occurrence is independent of its meaning, or any other substituent""s meaning, at any other occurrence, unless specified otherwise.
Suitably R8 is xe2x80x94ORxe2x80x2, xe2x80x94OCRxe2x80x22C(O)OR, xe2x80x94OCRxe2x80x22C(O)NRxe2x80x22 or OCRxe2x80x22C(O)xe2x80x94Rxe2x80x2, preferably xe2x80x94ORxe2x80x2.
Suitably R5, R10 and R11 are H.
Suitably (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94V is (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94 or xe2x80x94Uxe2x80x94(CRxe2x80x22)s.
Suitably U is CO, CONRxe2x80x2 or NRxe2x80x2CO.
Preferably, (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94V is (CH2)0-2NRxe2x80x2CO, (CH2)0-2CONRxe2x80x2, (CH2)0-2CO, (CH2)0-2CHxe2x95x90CH, (CH2)0-2Cxe2x89xa1C, (CH2)1-3O, or (CH2)1-5. More preferably (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94V is (CH2)0-2NRxe2x80x2CO or (CH2)0-2CONRxe2x80x2, where Rxe2x80x2 is H or methyl.
Preferably W is 
Preferably, Z is piperidinyl, piperazinyl or (CH2)t. Suitably t is 1 or 2.
Particular examples of R6 are: 
and Rxe2x80x3HNxe2x80x94(CH2)5xe2x80x94U wherein E is N or CH, R20 is hydrogen, amino, mono or di-C1-4alkylamino, hydroxy or C1-4alkyl, and U is NRxe2x80x2CO, CONRxe2x80x2, (CH2)CO, CHxe2x95x90CH, Cxe2x89xa1C, CH2O, OCH2 and (CH2)2.
Preferred illustrative examples of R6 are: 
wherein Rxe2x80x2 and Rxe2x80x3 are H or C1-4alkyl. Preferably Rxe2x80x2 is methyl and Rxe2x80x3 is H.
In a preferred embodiment, A1 is NR1, and A4 is NRxe2x80x2.
In another preferred embodiment, A1 is CHR1, and A4 is NRxe2x80x2.
Preferably, R2 is CH2xe2x80x94R7. More preferably, R2 is CH2CO2Rxe2x80x2, particularly CH2CO2H.
Preferably, R1 and R4 are H, C1-4alkyl, Arxe2x80x94C1-4alkyl or C3-6cycloalkyl-C1-4alkyl. Suitably R1 is H or methyl and R4 is H, methyl, cyclohexylethyl or phenylethyl.
In a more specific preferred embodiment, A1 is NRxe2x80x3 or CH2, where Rxe2x80x3 is H, C1-4alkyl or C(O)Rxe2x80x2; R2 is CH2CO2H; A4 is NR4; R4 is H, C1-6alkyl, C3-6cycloalkyl-C0-4alkyl or Arxe2x80x94C0-4alkyl; Z is a six-membered Het or (CH2)t; W is Rxe2x80x22N, or ; (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94V is (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94 or xe2x80x94Uxe2x80x94(CRxe2x80x22)s, (e.g., V is absent and one of s and r are 0) wherein U is NRxe2x80x2CO, CONRxe2x80x2, CRxe2x80x2xe2x95x90CRxe2x80x2, Cxe2x89xa1C, O, CO or CH2. Suitably, (CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94V is (CH2)0-3NRxe2x80x2CO, (CH2)0-3CONRxe2x80x2, where Rxe2x80x2 is H or methyl, or (CH2)0-3O. Preferably Rxe2x80x2 is methyl.
Specific embodiments of this invention, including useful intermediates and prodrugs, are described in Examples 1-88 inclusive.
In the above description of formula (I), W represents a nitrogen-containing group which is capable of making a hydrogen bond. Preferably W is a basic nitrogen moiety. R7 represents a group with a non-bonding pair of electrons which is capable of forming a hydrogen bond or chelating with a metal cation. Preferably R7 is acidic. It is also preferred that 10-15 (most preferably about 13) intervening covalent bonds via the shortest intramolecular path will exist between the group R7 and a terminal basic nitrogen moiety of W for optimal spacing between these groups, and the moieties T, U, V and Z, and the alkyl spacers represented by q, r, s are chosen accordingly. For instance, by way of illustration, but not limitation, when one of R2 or R4 is (CH2)2CO2H, or preferably CH2CO2H, and R6 is a substituent in the 7- or 8-position of the benzodiazepine ring system and is Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(e.g., s is 0 and V is absent), then: when W is  (preferably a 4-substituted six-membered nitrogen heterocycle), and Z is (CH2)t, and U is chosen from NRxe2x80x2CO, CONRxe2x80x2, CH2O, OCH2, CH2CH2, CRxe2x80x2xe2x95x90CRxe2x80x2 or Cxe2x89xa1C, (xe2x80x98group 1xe2x80x99), suitably q+t+r is 1-3 and preferably q+t+r is 1; when W is  and Z is a six-membered Ar or Het ring (preferably 1,4-disubstituted), and U is O, CH2 or CO, q and r are preferably 0; when W is H2Nxe2x80x94 and Z is (CH2)t, and U is chosen from group 1 above, q+r+t is 4-6, preferably 5; when W is H2Nxe2x80x94 and Z is a six-membered Ar or Het ring, suitably q+t is 0-2, preferably 1.
Abbreviations and symbols commonly used in the peptide and chemical arts are used herein to describe the compounds of this invention. In general, the amino acid abbreviations follow the IUPAC-IUB Joint Commission on Biochemical Nomenclature as described in Eur. J. Biochem., 158, 9 (1984).
Arg refers to arginine, MeArg refers to Nxcex1-methyl-arginine, HArg refers to homoarginine, NArg refers to norarginine, (Me2)Arg refers to Nxe2x80x2,Nxe2x80x3-dimethyl arginine, (Et2)Arg refers to Nxe2x80x2,Nxe2x80x3-diethyl arginine and Orn refers to ornithine. These radicals are suitable components of the substituent R6. Nxcex1-Substituted derivatives of these amino acid are also useful in this invention. Representative methods for preparing a-substituted derivatives are disclosed in U.S. Pat. No. 4,687,758; Cheung et al., Can. J. Chem., 55, 906 (1977); Freidinger et al., J. Org. Chem., 48, 77, (1982); and Shuman et al., Peptides: Proceedings of the 7th American Peptide Symposium, Rich, D., Gross, E., Eds, Pierce Chemical Co., Rockford, Ill.,617 (1981), which are incorporated herein by reference.
C1-4alkyl as applied herein is meant to include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl. C1-6alkyl additionally includes pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. C0-4alkyl and C0-6alkyl additionally indicates that no alkyl group need be present (e.g., that a covalent bond is present).
C2-6alkenyl as applied herein means an alkyl group of 2 to 6 carbons wherein a carbon-carbon single bond is replaced by a carbon-carbon double bond. C2-6alkenyl includes ethylene, 1-propene, 2-propene, 1-butene, 2-butene, isobutene and the several isomeric pentenes and hexenes. Both cis and trans isomers are included.
C2-6 alkynyl means an alkyl group of 2 to 6 carbons wherein one carbon-carbon single bond is replaced by a carbon-carbon triple bond. C2-6 alkynyl includes acetylene, 1-propyne, 2-propyne, 1-butyne, 2-butyne, 3-butyne and the simple isomers of pentyne and hexyne.
C1-4oxoalkyl refers to an alkyl group of up to four carbons wherein a CH2 group is replaced by a C(O), or carbonyl, group. Substituted formyl, acetyl, 1-propanal, 2-propanone, 3-propanal, 2-butanone, 3-butanone, 1- and 4-butanal groups are representative. C1-6oxoalkyl includes additionally the higher analogues and isomers of five and six carbons substituted by a carbonyl group. C3-6oxoalkenyl and C3-6oxoalkynyl refers to a C3-6alkenyl or C3-6alkynyl group wherein a CH2 group is replaced by C(O) group. C3-4oxoalkenyl includes 1-oxo-2-propenyl, 3-oxo-1-propenyl, 2-oxo-3-butenyl and the like.
A substituent on a C1-6alkyl, C2-6alkenyl, C2-6alkynyl or C1-6oxoalkyl group, such as R11, may be on any carbon atom which results in a stable structure, and is available by conventional synthetic techniques.
Qxe2x80x94C1-6 alkyl refers to a C1-6 alkyl group wherein in any position a carbon-hydrogen bond is replaced by a carbon-Q bond. Qxe2x80x94C2-6 alkenyl and Qxe2x80x94C2-6 alkynyl have a similar meaning with respect to C2-6 alkenyl and C2-6 alkynyl.
Ar, or aryl, as applied herein, means phenyl or naphthyl, or phenyl or naphthyl substituted by one to three moieties R11. In particular, R11 may be C1-4alkyl, C1-4alkoxy, C1-4alkylthio, trifluoroalkyl, OH, F, Cl, Br or I.
Het, or heterocycle, indicates an optionally substituted five or six membered monocyclic ring, or a nine or ten-membered bicyclic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by conventional chemical synthesis. Illustrative heterocycles are benzofuryl, benzimidazole, benzopyran, benzothiophene, furan, imidazole, indoline, morpholine, piperidine, piperazine, pyrrole, pyrrolidine, tetrahydropyridine, pyridine, thiazole, thiophene, quinoline, isoquinoline, and tetra- and perhydro- quinoline and isoquinoline. A six membered ring heterocycle containing one or two nitrogens, such as piperidine, piperazine, tetrahydropyridine and pyridine, are preferred heterocycles for the moiety Z. Any accessible combination of up to three substituents, such as chosen from R11, on the Het ring that is available by chemical synthesis and is stable is within the scope of this invention. A six membered monocyclic ring heterocycle containing one or two nitrogens, such as piperidine, piperazine, tetrahydropyridine and pyridine, are preferred heterocycles for the moiety Z.
C3-7cycloalkyl refers to an optionally substituted carbocyclic system of three to seven carbon atoms, which may contain up to two unsaturated carbon-carbon bonds. Typical of C3-7cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and cycloheptyl. Any combination of up to three substituents, such as chosen from R11, on the cycloalkyl ring that is available by conventional chemical synthesis and is stable, is within the scope of this invention.
 as used herein indicates a nitrogen heterocycle, which may be a saturated or unsaturated stable five-, six- or seven-membered monocyclic ring, or a seven- to ten-membered bicyclic ring containing up to three nitrogen atoms or containing one nitrogen atom and a heteroatom chosen from oxygen and sulfur, and which may be substituted on any atom that results in a stable structure. The nitrogen atom in such ring may be substituted so as to result in a quaternary nitrogen. The nitrogen heterocycle may be substituted in any stable position by R20, for instance H, C1-4alkoxy, F, Cl, Br, I, NO2, NRxe2x80x22, OH, CO2Rxe2x80x2, CONHRxe2x80x2, CF3, Qxe2x80x94C0-4alkyl, Qxe2x80x94C1-4alkyl-S(O)u (e.g., where u is 0, 1 or 2) or C1-4alkyl substituted by any of the aforementioned substituents. Representative of  are pyrroline, pyrrolidine, imidazole, imidazoline, imidazolidine, pyrazole, pyrazoline, pyrazolidine, piperidine, piperazine, morpholine, pyridine, pyridinium, tetrahydropyridine, tetrahydro- and hexahydro-azepine, quinuclidine, quinuclidinium, quinoline, isoquinoline, and tetra- and perhydro-quinoline and isoquinoline. In particular,  may be pyridyl, pyrolidinyl, piperidinyl, piperazinyl, azetidinyl, quinuclidinyl or tetrahydropyridinyl.  is preferably 4-pyridyl, 4-(2-amino-pyridyl), 4-tetrahydropyridyl, 4-piperidinyl or 4-piperazinyl.
AA1 as referred to herein is an amino acid with its carboxyl group optionally protected, wherein the amino acid may be any of the natural xcex1-amino acids or penicillamine. The unprotected carboxyl group is a free carboxylic acid group. Protecting groups for the carboxyl are esters or amides which are formed, for instance, when the OH of the carboxy group is replaced by R8. AA2 is an amino acid, as above, with its amino group optionally protected. Amino protecting groups are well known in the art, for instance, when the amino group is substituted by R12. An unprotected amino group is a free NH2 group.
C(O) indicates a carbon doubly bonded to oxygen (e.g., carbonyl), C(S) indicates a carbon doubly bonded to sulfur (e.g., thiocarbonyl).
t-Bu refers to the tertiary butyl radical, Boc refers to the t-butyloxycarbonyl radical, Fmoc refers to the fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Cbz refers to the benzyloxycarbonyl radical, BrZ refers to the o-bromobenzyloxycarbonyl radical, ClZ refers to the o-chlorobenzyloxycarbonyl radical, Bzl refers to the benzyl radical, 4-MBzl refers to the 4-methyl benzyl radical, Me refers to methyl, Et refers to ethyl, Ac refers to acetyl, Alk refers to C1-4alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to cyclohexyl. Tet refers to 5-tetrazolyl.
DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DEA refers to diisopropylethyl amine, EDC refers to N-ethyl-Nxe2x80x2(dimethylaminopropyl)-carbodiimide.HOBt refers to 1-hydroxybenzotriazole, THF refers to tetrahydrofuran, DIEA refers to diisopropylethylamine, DMF refers to dimethyl formamide, NBS refers to N-bromo-succinimide, Pd/C refers to a palladium on carbon catalyst, PPA refers to 1-propanephosphonic acid cyclic anhydride, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid, TEA refers to triethylamine, TFA refers to trifluoroacetic acid, PCC refers to pyridinium chlorochromate.
The compounds of formula (I) are generally prepared by reacting a compound of the formula (XI) with a compound of the formula (XII): 
wherein
A1, A4, U, V, Rxe2x80x2, R10, s and r are as defined in formula (I), with any reactive functional groups protected;
L1 and L2 are functional groups which are capable of reacting to form the linkage xe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94;
R6xe2x80x3 is Wxe2x80x2xe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94 and any portion of the group xe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94 which is connected to L2, with any reactive functional groups protected;
R2xe2x80x2 is R2 as defined for formula (I) with any reactive group protected; and
Wxe2x80x2 is W as defined for formula (I) with any basic nitrogen group protected; to form a compound of the formula: 
xe2x80x83wherein R6xe2x80x2 is Wxe2x80x2xe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94;
and thereafter removing any protecting groups, and optionally forming a pharmaceutically acceptable salt.
It will be apparent that the precise identity of L1 and L2 will be dependent upon the site of the linkage being formed. General methods for preparing the linkage xe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94 are described, for example, in EP-A 0 372 486 and EP-A 0 381 033 and EP-A 0 478 363, which are incorporated herein by reference.
For instance, if V is CONH, L1 may be xe2x80x94NH2, L2 may be OH (as in an acid) or Cl (as in an acid chloride), and R6xe2x80x3 may be Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94C(O), with any functional groups optionally protected. For example, R6xe2x80x3 may be (benzyloxycarbonyl-amidino)benzoyl- or (Nxcex1Boc,Nguan-Tos)arginyl-. When L2 is OH, a coupling agent is used.
Similarly, if V is NHCO, L1 may be xe2x80x94CO2H or COxe2x80x94Cl, L2 may be xe2x80x94NH2, and R6xe2x80x3 may be Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94. For example, R6xe2x80x3 may be (benzyloxycarbonylamidino)-phenyl, (benzyloxycarbonylamino)methylbenzyl- or 6-(benzyloxycarbonylamino)hexyl-.
Where V is NHSO2, L1 may be SO2Cl, L2 may be xe2x80x94NH2 and R6xe2x80x3 may be as above. Where V is SO2NH, L1 may be xe2x80x94NH2 and L2 may be SO2Cl. Methods to prepare such sulfonyl chlorides are disclosed, for instance, in J. Org. Chem., 23, 1257 (1958).
If V is CHxe2x95x90CH, L1 may be xe2x80x94CHO, L2 may be CHxe2x95x90Pxe2x80x94Ph3 and R6xe2x80x3 may be Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94. Alternately, L1 may be CHxe2x95x90Pxe2x80x94Ph3, L2 may be CHO, e.g., R6xe2x80x3 may be Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x88x921xe2x80x94CHO.
Where V is CH2CH2, compounds may be obtained by reduction of a suitably protected compound wherein V is CHxe2x95x90CH.
Where V is CH2O, CH2N or Cxe2x89xa1C, L1 may be xe2x80x94OH, xe2x80x94NH or xe2x80x94Cxe2x89xa1C H, respectively; L2 may be xe2x80x94Br, and R6xe2x80x3 may be Wxe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94. For example, R6xe2x80x3 may be (benzyloxycarbonylamino)-methylbenzyl- or 2-(N-benzyl-4-piperidinyl)-ethyl. Similarly where U or V is OCH2, NRxe2x80x2CH2 or Cxe2x89xa1C, L1 may be xe2x80x94CH2Br and L2 may be xe2x80x94OH, xe2x80x94NH or xe2x80x94Cxe2x89xa1C H, respectively. Alternately, when U or V is Cxe2x89xa1C, L1 may be Br, I or CF3SO3, L2 may be Cxe2x89xa1C H and the coupling may be catalyzed by palladium and a base.
Compounds wherein V is CHOHCH2 may be prepared from a suitably protected compound where V is CHxe2x95x90CH by the procedure disclosed in J. Org. Chem., 54, 1354 (1989).
Compounds wherein V is CH2CHOH may be obtained from a suitably protected compound where V is CHxe2x95x90CH by hydroboration and basic oxidation as disclosed in Tet. Lett., 31, 231 (1990).
The compounds of formula (XI), are benzodiazepines and benzazepines and are prepared by the general methods illustrated by Schemes 1-9. Representative methods for preparing benzodiazepines are well known in the art (e.g., Hynes, et al., J. Het. Chem., 1988, 25, 1173; Muller, et al., Helv. Chim. Acta., 1982, 65, 2118; Mori, et al., Heterocycles, 1981, 16, 1491); and WO 93/00095 which are incorporated herein by reference. Scheme 10 is illustrative of a method to prepare benzothiazepines. Benzoxazepines may be prepared in an analogous manner by starting with the compound wherein S is replaced by O. In the Schemes, R1xe2x80x3-R7xe2x80x3 indicate R1-R7 or a suitable precursor thereof, wherein any functional groups are protected as known in the art. 
A particularly useful intermediate is the 1,4-benzodiazepine compound of formula (XI), wherein A1 is NR1, A4 is NR4; L1 is CHO, CO2Rxe2x80x2, Br, I, OH, CF3SO3, CH2xe2x80x94T or NRxe2x80x2Rxe2x80x3, and T is OH, NHRxe2x80x3, Cl, Br or I. In particular, compounds wherein R1 is H, C1-4alkyl, C1-4oxoalkyl; R2 is CH2CO2Rxe2x80x2 and R4 is Qxe2x80x94C1-6alkyl are useful. More particularly, compounds wherein R4 is H, C1-4alkyl or phenylC1-4alkyl are useful. Other useful intermediates are similarly substituted benzazepine compounds of formula (XI), where A1 is CHR1 and A4 is NR4.
Coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling methods employ carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical.
Coupling methods to form peptide bonds are generally well known to the art The methods of peptide synthesis generally set forth by Bodansky et al., The Practive of Peptide Synthesis, Springer-Verlag, Berlin, 1984, Ali et al. in J. Med. Chem., 29, 984 (1986) and J. Med. Chem., 30, 2291 (1987) are generally illustrative of the technique and are incorporated herein by reference.
Solution synthesis for the formation of amide or peptide bonds is accomplished using conventional methods used to form amide bonds. Typically, the amine or aniline is coupled via its free amino group to an appropriate carboxylic acis substrate using a suitable carbodiimide coupling agent, such as N,Nxe2x80x2 dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole (HOBt) and dimethylamino pyridine (DMAP). Other methods, such as the formation of activated esters, anhydrides or acid halides, of the free carboxyl of a suitably protected acid substrate, and subsequent reaction with the free amine of a suitably protected amine, optionally in the presence of a base, are also suitable. For example, a protected Boc-amino acid or Cbz-amidino benzoic acid is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran(THF), in the presence of a base, such as N-methyl morpholine, DMAP or a trialkylamine, with isobutyl chloroformate to form the xe2x80x9cactivated anhydridexe2x80x9d, which is subsequently reacted with the free amine of a second protected amino acid or aniline.
Compounds of formula (XII) are prepared by conventional methods known in the art from commercially available materials. W is a generally a basic functional group attached to Z, optionally via an alkyl chain, and is protected during the synthesis of R6 or is introduced into the molecule after the xe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94Vxe2x80x94 linkage has been formed. For example, compounds of formula (XII) or formula (I) wherein W is a suitably substituted Rxe2x80x2Rxe2x80x3Nxe2x80x94, Rxe2x80x3Rxe2x80x2NC(xe2x95x90NRxe2x80x2), Rxe2x80x22N(R13)Cxe2x95x90Nxe2x80x94, Rxe2x80x3Nxe2x95x90(R13)Cxe2x80x94NRxe2x80x2xe2x80x94, Rxe2x80x22N(Rxe2x80x22N)Cxe2x95x90Nxe2x80x94 or Rxe2x80x3Rxe2x80x2N(Rxe2x80x2Nxe2x95x90)Cxe2x80x94NRxe2x80x2, are prepared by conventional methods including those disclosed in EP-A 0 372 486, EP-A 0 381 033 or EP-A 0 478 363, which are incorporated herein by reference.
Compounds of formula (XII) wherein W is  are prepared, inter alia, by methods disclosed in EP-A 0 478 363.
Compounds wherein W is Rxe2x80x22N(Rxe2x80x22N)Cxe2x95x90Nxe2x80x94Xxe2x80x94 or Rxe2x80x3Rxe2x80x2N(Rxe2x80x2Nxe2x95x90)Cxe2x80x94NRxe2x80x2xe2x80x94Xxe2x80x94, and X is O are prepared, inter alia, by methods disclosed in J. Org. Chem., 51, 5047 (1986).
Compounds wherein W is Rxe2x80x22N(Rxe2x80x22N)Cxe2x95x90Nxe2x80x94Xxe2x80x94 or Rxe2x80x3Rxe2x80x2N(Rxe2x80x2Nxe2x95x90)Cxe2x80x94NRxe2x80x2xe2x80x94Xxe2x80x94, and X is Nxe2x95x90CRxe2x80x2, are prepared, inter alia, by methods disclosed in U.S. Pat. No. 3,714,253 and Eur. J. Med. Chem.-Chim. Ther., 20, 25 (1985).
Compounds wherein W is Rxe2x80x22N(Rxe2x80x22N)Cxe2x95x90Nxe2x80x94Xxe2x80x94 or Rxe2x80x3Rxe2x80x2N(Rxe2x80x2Nxe2x95x90)Cxe2x80x94NRxe2x80x2xe2x80x94Xxe2x80x94, and X is C(O), are prepared, inter alia, by methods disclosed in U.S. Pat. No. 3,714,253 and Can. J. Chem., 43, 3103 (1965).
Compounds wherein W is Rxe2x80x2ONRxe2x80x2C(xe2x95x90NRxe2x80x2)xe2x80x94 may be prepared, inter alia, by methods disclosed in J. Het. Chem., 16, 1063 (1979) or J. Het. Chem., 26, 125 (1989).
Compounds wherein W is Rxe2x80x22NRxe2x80x2NC(xe2x95x90NRxe2x80x2)xe2x80x94 are prepared by conventional methods including those disclosed in Synthesis, 583 (1974).
Compounds wherein W is Rxe2x80x2Rxe2x80x3NRxe2x80x2Nxe2x80x94 are prepared, inter alia, by methods disclosed in J. Prakt. Chem., 36, 29 (1967).
Compounds wherein W is Rxe2x80x2Rxe2x80x3NRxe2x80x2NCOxe2x80x94 are prepared, inter alia, by methods disclosed in Bull. Chem. Soc. Jpn., 43, 2257 (1970).
Compounds wherein W is Rxe2x80x3Rxe2x80x2NC(xe2x95x90NRxe2x80x2)Y, and Y is S, are prepared, inter alia, by methods disclosed in Chem. Lett., 1379 (1986).
Compounds of formula (XII) or formula (I), wherein W is Rxe2x80x3Rxe2x80x2NC(xe2x95x90NRxe2x80x2)Y and Y is O, are prepared by conventional methods including those disclosed in Japanese Patent 2022751.
Useful intermediates of formula (XII) include compounds of the formula Wxe2x80x2xe2x80x94(CRxe2x80x22)qxe2x80x94Zxe2x80x94(CRxe2x80x2R10)rxe2x80x94Uxe2x80x94(CRxe2x80x22)sxe2x80x94L2, wherein Z, Rxe2x80x2, Rxe2x80x3, R10, U, q, r, and s are as defined for formula (I); L2 is CHO, CO2Rxe2x80x2, Cxe2x89xa1Cxe2x80x94H, OH, Cl, Br, I, CH2xe2x80x94T or NRxe2x80x2Rxe2x80x3, and T is CF3SO3, OH, NHRxe2x80x3, Cl, Br or I; and Wxe2x80x2 is W with any reactive basic nitrogen group protected as herein described by Rp, a nitrogen protecting group. Rxe2x80x2SO2, Rxe2x80x2OCO and Rxe2x80x2CO (e.g., Tos, Boc, Cbz or acetyl) are typical nitrogen protecting groups. Particular examples of such intermediates are: 
wherein E is N or CH, R20 is hydrogen, amino, mono or di-C1-4alkylamino, hydroxy or C1-4alkyl.
The reactive functional groups of the sidechains of each synthetic fragment are suitably protected as known in the art. Suitable protective groups are disclosed in Greene, Protective Groups in Organic Chemistry, John Wiley and Sons, New York, 1981. For example, the Boc, Cbz, phthaloyl or Fmoc group may be used for protection of an amino or amidino group. The Boc group is generally preferred for protection of an xcex1-amino group. A t-Bu, cHex or benzyl ester may be used for the protection of the side chain carboxyl. A benzyl group or suitably substituted benzyl group (e.g., 4-methoxy-benzyl or 2,4-dimethoxy-benzyl) is used to protect the mercapto group or the hydroxyl group. The tosyl group may be used for protection of the imidazolyl group and tosyl or nitro group for protection of the guanidino group. A suitably substituted carbobenzyloxy group or benzyl group may be also be used for the hydroxyl group or amino group. Suitable substitution of the carbobenzyloxy or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group. Except for the Boc group, the protective groups for the amino moiety are, most conveniently, those which are not removed by mild acid treatment These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment, as known in the art.
Modification of amino groups especially on the six-membered ring of the bicyclic system, may be accomplished by alkylation, sulfonylation, cyanation or acylation as is generally known in the art.
Acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic. The acetate salt form is especially useful. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts.
This invention provides a pharmaceutical composition which comprises a compound according to formula (I) and a pharmaceutically acceptable carrier. Accordingly, the compounds of formula (I) may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of formula (I) prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these peptides may be encapsulated, tableted or prepared in a emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the peptides of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
The compounds of this invention may be used in vitro to inhibit the aggregation of platelets in blood and blood products, e.g., for storage, or for ex vivo manipulations such as in diagnostic or research use.
This invention also provides a method of inhibiting platelet aggregation and clot formation in a mammal, especially a human, which comprises the internal administration of a peptide of formula (I) and a pharmaceutically acceptable carrier. Indications for such therapy include acute myocardial infarction (AMI), deep vein thrombosis, pulmonary embolism, dissecting anurysm, transient ischemia attack (TIA), stroke and other infarct-related disorders, and unstable angina. Chronic or acute states of hyper-aggregability, such as disseminated intravascular coagulation (DIC), septicemia, surgical or infectious shock, post-operative and post-partum trauma, cardiopulmonary bypass surgery, incompatible blood transfusion, abruptio placenta, thrombotic thrombocytopenic purpura (TTP), snake venom and immune diseases, are likely to be responsive to such treatment. In addition, the peptides of this invention may be useful in a method for the prevention of metastatic conditions, the prevention or treatment of fungal or bacterial infection, inducing immunostimulation, treatment of sickle cell disease, and the prevention or treatment of diseases in which bone resorption is a factor.
The peptide is administered either orally or parenterally to the patient, in a manner such that the concentration of drug in the plasma is sufficient to inhibit platelet aggregation, or other such indication. The pharmaceutical composition containing the peptide is administered at a dose between about 0.2 to about 50 mg/kg in a manner consistent with the condition of the patient. For acute therapy, parenteral administration is preferred. For persistent states of hyperaggregability, an intravenous infusion of the peptide in 5% dextrose in water or normal saline is most effective, although an intramuscular bolus injection may be sufficient.
For chronic, but noncritical, states of platelet aggregability, oral administration of a capsule or tablet, or a bolus intramuscular injection is suitable. The peptide is administered one to four times daily at a level of about 0.4 to about 50 mg/kg to achieve a total daily dose of about 0.4 to about 200 mg/kg/day.
This invention further provides a method for inhibiting the reocclusion of an artery or vein following fibrinolytic therapy, which comprises internal administration of a peptide of formula (I) and a fibrinolytic agent. It has been found that administration of an peptide in fibrinolytic therapy either prevents reocclusion completely or prolongs the time to reocclusion.
When used in the context of this invention the term fibrinolytic agent is intended to mean any compound, whether a natural or synthetic product, which directly or indirectly causes the lysis of a fibrin clot. Plasminogen activators are a well known group of fibrinolytic agents. Useful plasminogen activators include, for example, anistreplase, urokinase (UK), pro-urokinase (pUK), streptokinase (SK), tissue plasminogen activator (tPA) and mutants, or variants, thereof, which retain plasminogen activator activity, such as variants which have been chemically modified or in which one or more amino acids have been added, deleted or substituted or in which one or more or functional domains have been added, deleted or altered such as by combining the active site of one plasminogen activator with the fibrin binding domain of another plasminogen activator or fibrin binding molecule. Other illustrative variants include tPA molecules in which one or more glycosylation sites have been altered. Preferred among plasminogen activators are variants of tPA in which the primary amino acid sequence has been altered in the growth factor domain so as to increase the serum half-life of the plasminogen activator. tPA Growth factor variants are disclosed, e.g., by Robinson et al., EP-A 0 297 589 and Browne et al., EP-A 0 240 334. Other variants include hybrid proteins, such as those disclosed in EP 0 028 489, EP 0 155 387 and EP 0 297 882, all of which are incorporated herein by reference. Anistreplase is a preferred hybrid protein for use in this invention. Fibrinolytic agents may be isolated from natural sources, but are commonly produced by traditional methods of genetic engineering.
Useful formulations of tPA, SK, UK and pUK are disclosed, for example, in EP-A 0 211 592, EP-A 0 092 182 and U.S. Pat. No. 4,568,543, all of which are incorporated herein by reference. Typically the fibrinolytic agent may be formulated in an aqueous, buffered, isotonic solution, such as sodium or ammonium acetate or adipate buffered at pH 3.5 to 5.5. Additional excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene, glycol, mannitol and sodium chloride may also be added. Such a composition can be lyophilized.
The pharmaceutical composition may be formulated with both the compound of formula (I) and fibrinolytic in the same container, but formulation in different containers is preferred. When both agents are provided in solution form they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
Indications for such therapy include myocardial infarction, deep vein thrombosis, pulmonary embolism, stroke and other infarct-related disorders. The peptide is administered just prior to, at the same time as, or just after parenteral administration of tPA or other fibrinolytic agent. It may prove desirable to continue treatment with the peptide for a period of time well after reperfusion has been established to maximally inhibit post-therapy reocclusion. The effective dose of tPA, SK, UK or pUK may be from 0.5 to 5 mg/kg and the effective dose of the peptide may be from about 0.1 to 25 mg/kg.
For convenient administration of the inhibitor and the fibrinolytic agent at the same or different times, a kit is prepared, comprising, in a single container, such as a box, carton or other container, individual bottles, bags, vials or other containers each having an effective amount of the inhibitor for parenteral administration, as described above, and an effective amount of tPA, or other fibrinolytic agent, for parenteral administration, as described above. Such kit can comprise, for example, both pharmaceutical agents in separate containers or the same container, optionally as lyophilized plugs, and containers of solutions for reconstitution. A variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use. With such an arrangement, the fibrinolytic and the peptide may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container.
When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement. For example, the platelet aggregation inhibitor may be in an i.v. injectable form, or infusion bag linked in series, via tubing, to the fibrinolytic agent in a second infusion bag. Using such a system, a patient can receive an initial bolus-type injection or infusion, of the peptide inhibitor followed by an infusion of the fibrinolytic agent.
The pharmacological activity of the compounds of this invention is assessed by their ability to inhibit the binding of 3H-SKandF 107260, a known RGD-fibrinogen antagonist, to the GPIIbIIIa receptor, their ability to inhibit platelet aggregation, in vitro, and their ability to inhibit thrombus formation in vivo.
Inhibition of RGD-mediated GPIIb-IIIa Binding
Inhibition of RGD-mediated GPIIb-IIIa binding was demonstrated by assessing the ability of compounds to inhibit the binding of 3H-SKandF 107260, a known RGD-fibrinogen antagonist, to the GPIIbIIIa receptor according to the procedure disclosed in WO 93/00095 (PCT/US/92/05463).
Inhibition of Platelet Aggregation
Inhibition of platelet aggregation was demonstrated following the procedure disclosed in WO 93/00095 (PCT/US/92/05463).
The compounds of this invention inhibit the aggregation of human platelets stimulated with ADP with IC50 of about 0.001 to about 150 xcexcM. Preferred compounds have IC50 of less than 0.1 xcexcM.
To assess the stability of the compounds to plasma proteases, the compounds were incubated for 3 h (rather than 3 min) in the PRP prior to addition of the agonist.
In Vivo Inhibition of Platelet Aggregation
In vivo inhibition of thrombus formation is demonstrated by recording the systemic and hemodynamic effects of infusion of the peptides into anesthetized dogs according to the methods described in Aiken et al., Prostaglandins, 19, 629 (1980).
The examples which follow are intended to in no way limit the scope of this invention, but are provided to illustrate how to make and use the compounds of this invention. Many other embodiments will be readily apparent and available to those skilled in the art.